RhoGDI Pathway
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RhoGDI Pathway

The Rho family of small GTPase proteins comprises CDC42 (Cell Division Cycle-42), Rac, and Rho. Proteins of the Rho/Rac subfamily (Rho proteins) of small GTP-binding proteins function as molecular switches that regulate a multitude of biological processes including cell proliferation, apoptosis, differentiation, migration, cytoskeletal reorganization, and membrane trafficking. Like most of the Ras-related proteins, Rho GTPases cycle between an inactive GDP-bound conformation and an active GTP- bound conformation. Cycling between the two conformations enables these proteins to act as binary switches (Ref.1). The tightly regulated GTP-binding/GTPase cycle requires the coordinated action of three regulatory proteins: 1) GEFs (Guanine Nucleotide Exchange Factors), which stimulate the GTP-GDP exchange reaction; (2) GAPs (GTPase-Activating Proteins), which stimulate the GTP-hydrolytic reaction; and (3) GDIs (Guanine Nucleotide Dissociation Inhibitors), which antagonize the actions of both GEFs and GAPs. Only three RhoGDIs have been described: RhoGDI1 (also called RhoGDI or RhoGDIa) (ii) RhoGDI2 (also named D4/Ly-GDI or RhoGDIb) and (iii) RhoGDI3 (also named GDIg). The ubiquitously expressed RhoGDI1 seems to have a broad action and is able to inhibit and to form tight 1: 1 cytosolic complexes with RhoA, Rac1, Rac2, and CDC42. PAK1 (p21-Activated Protein Kinase), a serine/threonine kinase that regulates the actin cytoskeleton, is an effector of the Rho family GTPases CDC42 and Rac1. In response to growth factors PI3K (Phosphatidylinositol-3 Kinases) in turn activates CDC42 and PAK. PAKs stimulated by binding with GTP-liganded forms of CDC42 or Rac, modulate cytoskeletal actin assembly and activate MAP Kinase pathways. RhoGDI2 is specifically expressed in hematopoietic tissues and predominantly in B- and T-lymphocyte cell lines. RhoGDI3 has a less ubiquitous expression than RhoGDI1. It is preferentially expressed in brain tissues but also found in lung, kidney, and testis. The most remarkable feature of RhoGDI3 is its association with a particulate subcellular fraction, in contrast with the other known RhoGDIs, which are only found in the cytosolic fraction. Thus, RhoGDI3 has a different action compared to cytosolic RhoGDIs. RhoGDI3 is the only member of the RhoGDI family known to be partially associated with a particulate subcellular fraction. Noncytosolic fraction of GDI3 is associated with the Golgi apparatus. Golgi-associated GDI3 protein regulates the RhoG activity and localization (Ref.2).

GDI has three activities 1) GDI keeps the GDP-bound form in the cytosol; 2) GDI transports its complexed small G-protein to its respective target membrane where the GDP-bound form is converted to the GTP-bound form; and 3) Once the GDP-bound form is converted from the GTP-bound form after its function has been accomplished, GDI forms a complex with it and translocates it to the cytosol. RhoGDIs interact preferentially with GDP-bound forms of Rho proteins preventing spontaneous GEF-catalyzed release of the GDP nucleotide, thereby maintaining the GTPases in an inactive state. In addition, RhoGDI controls membrane localization of the GTPases. Most proteins of the Rho family cycle between cytosol and membranes. Membrane association of Rho proteins is mediated by the insertion of the isoprenyl moiety, situated at their C-terminus, into the lipid bilayer. Transfer of the isoprenoid moiety from the lipid bilayer to a binding pocket within the GDI directly results in the dissociation of the CDC42/GDI complex from the membrane producing a soluble species (Ref.3).

Since the Rho GTPases promote cytoskeletal and membrane changes associated with apoptotic cell death, the removal of the GDI block through its cleavage is important for inducing apoptosis. Caspase3 cleaves the 28 kDa mature form of RhoGDI-Beta to give a 5 kDa and 23 kDa Size fragment. The 23 kDa fragment then translocates to the nucleus. Activation of the JNK (Jun N-Terminal kinase), a regulator of apoptosis, may be one of the mechanisms involving cleavage of GDI (Ref.4).

The RhoGDIs negatively regulate Rho-family GTPases. RhoGDI inhibits the functions of the Rho family members, such as cytokinesis, cell motility, membrane ruffling, and formation of stress fibers and focal adhesions. The inhibitory activity of GDI derives both from an ability to bind the carboxy-terminal isoprene of Rho family members and extract them from membranes and from inhibition of GTPase cycling between the GTP- and GDP-bound states. These binding and inhibitory functions of RhoGDI can be attributed to two structurally distinct regions of the protein. A carboxy-terminal folded domain of relative molecular mass 16,000 (16K) binds strongly to the Rho-family member CDC42, yet has little effect on the rate of nucleotide dissociation from the GTPase. The amino-terminal region of RhoGDI is unstructured in the absence of target and contributes little to binding, but is necessary to inhibit nucleotide dissociation from CDC42. These results lead to a model of RhoGDI function in which the carboxy-terminal binding domain targets the amino-terminal inhibitory region to GTPases, resulting in membrane extraction and inhibition of nucleotide cycling. Although RhoGDI is predominantly thought to be a negative regulator of Rho family G-proteins, recent work suggests a potential positive role for RhoGDI in Rho family signaling. RhoGDI was also found to associate with a protein complex containing ERM (Ezrin, Radixin, and Moesin) proteins and their membrane-binding partner, CD44. The interaction between ERM proteins and CD44, which appears to be regulated by Rho, is important for cross-linking the plasma membrane with actin filaments. The association of Rac with the PtdInsP 5-kinase and , DGK is GTP independent, and the majority of GDP-bound Rac in cells is present in a complex with RhoGDI. RhoGDI bound to both the DGK and the PtdInsP 5-kinase. This finding supports the idea that RhoGDI could have positive signaling functions (Ref.5).